Method of treating bacterial infections

ABSTRACT

AND ANTIBACTERIAL AMOUNT OF CERTAIN DIPETIDES OF HISTIDINE IS ADMINISTERED TO MAMMALS IN NEED TO AN ANTIMICROBIAL EFFECTIVE IN PROTECTING AGAINST COCCI AND BACILLI.

United States Patent METHOD OF TREATING BACTERIAL INFECTIONS Elton S. Cook and Kinji Tanaka, Cincinnati, Ohio, as-

signors to Stanley Drug Products, Inc., Portland, Oreg. No Drawing. Continuation-impart of abandoned application Ser. No. 697,002, Jan. 11, 1968. This application Dec. 4, 1969, Ser. No. 882,266

Int. Cl. A61k 27/00 US. Cl. 424-177 4 Claims ABSTRACT OF THE DISCLOSURE An antibacterial amount of certain dipeptides of histid ne is administered to mammals in need of an antimicrobial effective in protecting against cocci and bacilli.

BACKGROUND OF THE INVENTION This is a continuation-in-part of Ser. No. 697,002, filed Ian. 11, 1968, now abandoned. The invention pertains to antimicrobials. In a particular aspect this invention relates to antimicrobials effective in protecting against bacterial infections caused by cocci and bacilli.

Bacteria such as cocci and bacilli are organisms embodying within themselves and array of yet unanswered puzzles in biology, both fundamental and experimental. They are ubiquitous in distribution and have attained extreme degrees of diversification in biological and biochemical characteristics. It is recognized that the significance of staphylococcal infections is not so much in severity, except in a few instances, as in the subtleties of the infection due to the unpredictable vagaries of these organisms.

Treatment of staphylococcal diseases is complicated by the ability of the organisms to develop resistance. The magnitude of the problem is further amplified by the extreme difiiculty of total eradication, and the frequent reappearance of the same strain even after apparently successful elimination. The inability to eliminate the carrier state by any of the currently known methods and the prevalence of the new antibiotic resistant hospital strains have added a new dimension to the frustrating situation.

Penicillin G (benzyl penicillin) is still the drug of choice for the treatment of infections caused by susceptible coccic strains. However numerous strains are known which elaborate an enzyme penicillinase in response to the drug and thus remain insensitive. This led to the development of semi-synthetic penicillins which are not inactivated by penicillinase. However recently resistance of staphylococci to the newer penicillins has been reported. Hence there is a seemingly never ending demand for anticoccic factors.

A variety of substances are reported which alter host resistance to coccic infections. However, drugs which induce a high degree of resistance to coccic infections are not in wide use. In addition drugs which confer immunity to coccic infections for more than a week after administration are virtually unknown. In one of its aspects this invention provides an antimicrobial for the treatment of staphylococcic and bacillic infections. By another aspect of this invention is to give immunity to reinfection.

SUMMARY OF THE INVENTION The dipeptide of beta-alanine and histidine known as carnosine, and its homolog the dipeptide of gammaaminobutyric acid and histidine, known as homocarnosine, have been identified in the brains of mammals. No definite biochemical role has been ascribed to carnosine (beta-alanyl histidine) even though it has been known for over sixty years. Homocarnosine (gamma-aminobutyryl histidine) is relatively new. Obviously the physiological function of this dipeptide has not yet been ascertained.

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The presence of these compounds in the brain argues for a definite function. In accordance with this invention it has been found that homologs of carnosine afford extremely satisfatcory degrees of protection against cocci and bacilli infections. Thus homocarnosine possesses an anticoccic activity unshared by lower homologs. Carnosine, which differs from homocarnosine only by having one less carbon atom, is unsatisfactory in building resistance to coccic infections. According to another aspect of the invention two homologs of carnosine higher than homocarnosine are also desirable antimicrobials. These are also new compositions of matter, being the subject of copending application Ser. No. 831,741, filed June 9, 1969. Thus although the physiological function of homocarnosine is still speculative, and its higher homologs unknown, according to the practice of this invention, we have discovered its unique activity against bacterial infections.

DETAILED DESCRIPTION OF THE INVENTION The processes of infection leading to disease are accepted to be a problem in the ecology of the parasite. It is being increasingly realized that the bacterial and host determinants are closely interrelated. Staphylococcal virulence derives from the combined action of several bacterial factors whose effectiveness is conditioned by the reactions of the host. Perhaps the most striking feature of host-parasite relationships in staphylococcal infections is the relatively atypical immunologic response. Human studies have given convincing evidence that most adult humans possess an array of antibodies against staphylococci. Nevertheless resistance to staphylococci seems to be governed to a considerable extent by other unrelated factors. For this reason attempts at prophylactic administration have not been completely successful.

By the practice of this invention there is provided a method of protecting mammals against bacterial infections. In accordance with the invention an antibacterial amount of certain dipeptides of histidine is administered to mammals in need of an antimicrobial effective in protecting against cocci and bacilli. The dipeptides administered for the control of bacterial infections are gammaaminobutyrylhistidine (homocarnosine), and two new compounds, delta aminovalerylhistidine and epsilonaminocaproylhistidine. Whereas the dipeptide per se can be used, it will usually be administered as an edible salt such as delta-aminovaleric acid chloride, epsilon-aminocaproic acid phosphate, homocarnosine gluconate, homocarnosine lactate, homocarnosine salicylate, homocarnosine carbonate, homocarnosine fluoride, homocarnosine iodide and the like. The salt is administered to mammals in pharmaceutically effective amounts, in the range of 150 to 250 milligram doses. The salts are easier to purify than the free dipeptide, and hence are more readily available in this form. In addition the salts are much more water soluble and will have lower pH values than the dipeptides themselves.

The high degree of resistance to staphylococcal infections obtained by the prophylactic administration of homocarnosine will be first demonstrated. This will be apparent from its biological effects in in vivo studies.

Swiss albino mice, 12 to 14 Weeks old, and weighing approximately 20 to 25 grams each, were used in most of this work. Results showed that animals of either sex give comparable results and hence in these studies the animals were generally not screened according to sex.

The challenge organism was the penicillin-resistant Staphylococcus aureus (named Original), a strain maintained in our laboratories for years. It is preserved in the lyophilized state and stored at 0 C. and stock cultures were raised on Staphylococci medium no. Difco slants once in every 6 months. For testing, the inoculum was prepared from 24 hour cultures on said slants at 37 C. The cells were washed and suspended in physiological saline (TC Tyrode Solution, Difco). In contrast to conventional procedures, an LD 8090 instead of LD 50 was used in these investigations. This has been the practice in our laboratories in studies with staphylococci since lower dosages often fail to give adequate degrees of mortality. The LD 8090 was determined by injecting groups of mice subcutaneously in the groin area with different dilutions of the bacterial suspension and noting the mortality over a 5-day period.

Homocarnosine sulfate purchased from biochemicals manufacturers was used in all of the work reported herein. It was injected subcutaneously as a one percent solution in dosages indicated in each test. Investigations established a definite antistaphylococcal activity for homocarnosine. Because of its relative molecular simplicity and the commercial availability, an intensive study of this dipeptide was undertaken. To begin with, a series of prophylactic tests were done in which each animal was given 5 mg. of homocarnosine sulfate in 3 days by the subcutaneous route. On the 4th day (24 hr. after the last administration) the mice were challenged with Staph. aureus using control groups for comparison. Table 1 summarizes the results of 25 experiments in which the mortalities of the treated mice ranged from to 30 percent.

TABLE 1 Prophylactic subcutaneous Percent No. of No. of mortality expts. animals in days During these studies it was noticed that commercially available homocarnosine was not always homogeneous in biological activity and physical appearance. The best samples were found to lower mortalities to O to percent. Each sample was separately assayed before being used in animal tests.

Once the antistaphylococcal property was established, it was important to determine the appropriate dose for optimum results. In all tests thus far, an arbitrary dose of 5 mg. per animal was used. Three groups of animals were now treated prophylactically in 3 days by the subcutaneous route as before with 5 mg., 2.5 mg. and 1.5 mg. of homocarnosine sulfate per animal. The extent of protection given by the respective dose can be seen in the results after challenge reproduced in Table 2.

The Work set forth in Table 2 suggests that even with 1.5 mg. per animal prophylactically, the protection against subsequent challenge is comparable with that of higher doses used before. However, in view of the variations in results obtained with higher doses and the possibility of having a certain proportion of the sample in an inactive form, it was thought desirable to continue to use the 5 mg. dose in these investigations. Besides, there was no indication of any toxicity at this dose which further favored this dose for routine use.

In tests thus far, the animals were challenged within 24 hours after the termination of treatment and hence all the data refer only to the protection within this narrow range of time. It was of interest to ascertain whether the resistance induced by homocarnosine is short-lived or long-lasting. A group of 50 mice were treated with homocarnosine (5 mg. per animal) in 3 subcutaneous injections. They were divided into 5 groups and each group was challenged with Staph. aureus at weekly intervals, using a fresh set of controls each time. The first group was challenged 24 hours after the last treatment as before. Mortalities in each group were recorded over periods of 5 days (Table 3).

TABLE 3 Duration of protection: prophylactic Percent mortality in days post-challenge No. of

Group animals 1 2 3 4 5 Challenged 24 hours after treatment Control 10 70 70 70 80 Homocarnosine 10 O 0 0 10 10 Challenged 7 days after treatment Control... 10 70 80 80 80 Homocamosine 10 0 0 O 0 0 Challenged 14. days after treatment Control 10 7O 90 9O 90 J0 Homocarnosine 10 20 20 20 20 20 Challenged 21 days after treatment Control 10 60 70 70 70 70 Hornocarnoslne l0 0 30 3O 30 30 Challenged 28 days after treatment Control 9 67 78 78 78 78 Hornocarnosin 9 22 22 22 22 22 Although there is a slight drop in the degree of protection with the lapse of time, the results indicate that homocarnosine by itself is able to protect mice against experimental staphyloccal infections at least up to a period of 1 month. It is clear from Table 3 that even in the last group challenged 28 days after treatment, the degree of protection is comparable with results in many past experiments on immediate effects.

As a corollary to the observations on the duration of protection, it was of interest to ascertain the minimum time after administration, required for homocarnosine to express its activity. Fifty animals were treated with 5 mg. homocarnosine each, in one subcutaneous injection. They were divided into 5 groups of 10 each and successive groups were challenged at intervals of 1 hour using a different control group each time. A fresh inoculum from a 24 hour culture was also used each time. Mortality in each group was recorded and compared against that of the control group as can be seen in Table 4.

TABLE 4 Minimum time for expression Percent mortality in days postehallenge N o. of Group animals 1 2 3 4 5 Challenged 1 hour after treatment Control t 10 50 60 60 60 60 Homoearnosme 10 20 20 20 20 20 Challenged 2 hours after treatment Control 10 40 so 50 50 6g Homoearnosme 10 20 30 30 30 3 Challenged 3 hours after treatment Control 7 10 5O 60 60 60 60 Homocarnosine 1O 20 20 20 .20 .20

Challenged 4 hours after treatment Control 10 30 60 60 60 60 Homoearnosme 1O 10 10 20 40 40 Challenged 5 hours after treatment Control 1 10 5O 50 5U 5O 5 llonlocarnosine 10 1o 10 10 l0 l0 Although the group challenged at 4 hours shows some inconsistency, it is evident that even as early as 1 hour after treatment, homocarnosine is able to express its antistaphylococcal property. In fact, the degree of protection compares well with that after hours.

Staphylococcal infections are known to recur. It was, therefore, of interest to see whether the survivors from the lethal challenge after prophylactic administration of homocarnosine are immunt to rechallenge and if so to what extent. Rechallenge experiments were performed at varying intervals after the first challenge up to a maximum of 59 days, and it was consistently observed that the survivors were almost completely immune at least up to the periods covered, as set forth in Table 5.

TABLE 5 Survivors rechallenged 59 days after the first challenge Percent mortality in days The results of in vivo experiments prompted experiments to study any direct effects of homocarnosine on the infecting organism itself. This was thought to be of value in attempting to explain the mechanism of action of this peptide. Emphasis was mostly on effects on growth pigmentation and the biochemical characteristics like the fermentation of mannitol, liquefaction of gelatin and the production of coagulase and DNase. An aqueous solution of homocarnosine .(1 mg./ ml.) was prepared and for each test 1 ml. of this was added to 4 ml. of the growth medium which varied depending upon the specific test. For chromogenesis and coagulase SA 110 (Difco) was used while Phenol Red Mannitol Agar (Difco) and Nutrient Gelatin (Difco) served as the test media for mannitol fermentation and gelatin liquefaction, respectively. DNase Agar (Difco) was used for DNase production. From these tests it was found that incorporation of homocarnosine into a growth medium, does not in any way alter the basic characteristics of the organism, nor does it affect the rate of growth. Filter paper discs soaked in homocarnosine solution placed on plates inoculated with Staph. aureus do not show any inhibition or enhancement of growth in their vicinity. Even when the same clone was continuously exposed to homocarnosine by maintenance on nutrient agar containing homocarnosine sulfate (1 percent by weight) for 20 subcultures at intervals of 3 days each, no significant changes were noticed.

Since homocarnosine is a dipeptide of gamma-aminobutyric acid and L-histidine, it was decided to compare the activity of homocarnosine with both the components when administered in equirnolar ratios. The results of this work are recorded in Table 6.

TABLE 6 Homocarnosine and mixture of L-histidine and gamma-aminobutyric acid: prophylactic Percent mortality in days Dose per post-challenge mouse No. of

Group in M animals 1 2 3 4 5 Control 90 90 90 90 90 Gamma-aminobutyric acid L-histidine ++15 10 70 70 80 80 80 Homocarnosine 15 10 20 20 20 20 Percent mortality in days Dose per post-challenge animal No. of Group in mg. animals 1 2 3 4 5 Control- 10 50 80 L carnoslne 10 10 70 100 100 100 Homocarnos 5 10 2O 30 30 30 30 These data further confirmed a definite value of homocarnosine in protecting Swiss albino mice against experimental staphylococcal infections. An analysis of the results of several experiments by this testing procedure revealed that experimental groups of Swiss mice given L-carnosine were not protected against the infection. Carnosine shows some effectiveness in C3H mice. However as can be seen in Table 8 homocarnosine is nevertheless much more effective. Then animals were used in each group, tested against Staphylococcus aureus.

TABLE 8 Carnosineprophylactic-subcutaneous- 03H mice Percent mortality in days whereas there are some differences as a result of host differences, homocarnosine is very effective in both strains of mice.

The value of homocarnosine in protecting mice against staphylococcal infections having been definitely established, data showing the antistaphylococcal activities of delta-aminovalerylhistidine (DAV-his) and epsilonaminocaproylhistidine (EAC-his) will now be given. Homocarnosine is unique in its activity. As is apparent from the foregoing tables, the administration of homocarnosine induces resistance to staphylococcal infections at least up to a period of one month if not longer. Particularly significant is the complete immunity to reinfection of up to two months after an infection preceded by a homocarnosine treatment. This period is perhaps longer than any reported so far with any immunizing procedure of which we are aware. The two dipeptides delta-aminovalerylhistidine (DAV-his) and epsilonaminocaproylhistidine (EAC-his) also constitute significant new antimicrobials. The excellent protection conferred by these two dipeptides is illustrated by the data in Table 9. Here the drug was given in 2 equal doses, /2 given 2 hours before infection with Staphylococcus aureus and /2 given 4 hours after infetcion.

TABLE 9 Dipeptides-prophylacticsubcutaneous-Swiss mice Total Percent mortality at hrs.

dose, No. Drug mg. mice 48 72 96 5 8 25 50 50 50 10 90 90 90 90 5 10 30 30 3O 30 10 70 80 80 80 5 3 25 25 25 25 10 40 50 50 80 5 9 0 22 22 22 5 10 30 4O 40 40 10 70 90 90 90 5 10 30 30 3O 30 5 8 25 25 25 20 10 50 100 100 100 5 10 0 0 0 5 5 10 0 0 0 0 10 50 70 80 80 It can be seen that by the practice of this invention staphylococcal infection will be reduced by administering a pharmaceutically effective amount of the dipeptide internally to the animal suffering from the infection. Similar results are obtained in the case of bacilli infections as will now be illustrated in the case of Salmonella old received 5 mg. of homocarnosine sulfate subcutaneously for 3 or 5 consecutive days, a total of or mg. Two or three hours after the final dose of homocarnosine the mice were given a subcutaneous challenge of an LD dose of Salmonella typhi. The resulting mortalities at 72 hours after challenge are given in Table 10 which summarizes four experiments.

25 The compositions of this invention thus constitute significant new antimicrobials. It is contemplated that they will be taken prior to periods Where contact with staphylococci, streptococci or Salmonella typhi infections are likely, such as prior to entering a hospital. They can be taken orally in 250 to 500 mg. tablets or as injections of, say, 150 to 500 mg, The dipeptide per se or the sulfate, chloride or other edible salt thereof can be combined with an aqueous, vegetable oily, monoglyceride or diglyceride vehicle for injection, sodium chloride being used if necessary to render the solution isotonic. The solution will contain 0.1 to 5 percent, preferably 0.1 to 1.5 percent by weight, of the dipeptide or soluble salt thereof.

In the case of tablets the dipeptide may be preferred to the salt. Suitable colorants, adhesives, and lubricants will be incorporated in the tablets along with a solid pharmaceutical diluent, for instance, starches, lactose, sucrose and other pharmaceutical diluents. These tablets will contain 0.08 percent to 5 weight percent of the dipeptide, preferably 0.08 percent to 1.3 weight percent. Generally the same quantity will be used in all instances where quantities have been given, whether the dipeptide or the salt thereof is employed, Capsules can also be made. Oral administration is illustrated by the following results, set forth in Table 11. The dipeptide used was homocarnosine. Each animal (Swiss mice) received a total dose of 10 mg. of homocarnosine sulfate as an aqueous solution of 20 mg. Homocarnosine sulfate per ml. in water according to the schedule in the table. The control group received only the Staph. aureus injection.

A process is thus provided for the control of infections in humans and other mammals due to cocci and bacilli, which involves administering to the mammal suffering from the infection an effective amount of the dipeptide or the edible salt thereof. In addition variations and modifications will occur to those skilled in the art. Thus an inoculum can be made from the serum of mammals which have recovered from the infection, This is illustrated as follows.

Mice which had received a total of 5 mg. of homocarnosine. subcutaneously over a three day period were inoculated subcutaneously with an amount of S. aureus normally sufficient to kill 80-90 percent of the animals. Mice which recovered from the infection after this treatment were bled to death by orbital bleeding. All the blood was pooled in centrifuge tubes and allowed to clot for about 30 minutes. After loosening the clot it was centrifuged for 12 minutes at 1700 r.p.m. and the clear supernatant serum Was carefully drawn off with a sterile Pasteur pipette. The serum was sterilized by passage through sterile millipore filters and stored in serum bottles at 10 C. for not more than 10 days.

All experimental group of 14'mice were each given 0.2 ml. of serum intravenously through the tail vein. A control group of 10 mice received the same volume of physiological saline solution All mice were challenged subcutaneously 24 hours later with a lethal dose of S. aureus. The results are shown in Table 12.

TABLE 12 Serum Percent mortality at days post-challenge It will be seen that the serum protected nearly half of the mice after one day and about a third of the mice over the five-day period, This effectiveness will be improved by further studies of variables such as dose of homocarnosine, pattern of homocarnosine dosage, dosage of serum, time interval between serum administration and challenge infection, and stability of serum. Such ramifications are deemed to be within the scope of this invention.

What is claimed is:

1. A method of treating bacterial infections in mammals comprising administering to a mammal suffering for said bacterial infection an antibacterial effective amount of a dipeptide selected from the group consisting of gamma-aminobutyrylhistidine, delta-aminovalerylhistidine and epsilonaminocaproylhistidine.

2. The method of claim 1 wherein the dipeptide is gamma-aminobutyrylhistidine.

3. The method of claim 1 wherein the dipeptide is delta-aminovalerylhistidine.

4. The method of claim 1 wherein the dipeptide is epsilon-aminocaproylhistidine.

References Cited Chemical Abstracts, :3395(c) (1964). Chemical Abstracts, 61:16397(b) (1964).

JEROME D. GOLDBERG, Primary Examiner 

